Figure 7. Caveolin Is Recruited to the C. jejuni–Containing Vacuole and Is Required for Efficient C. jejuni Entry Into Epithelial Cells.

Cos-1 cells were transfected with plasmids expressing caveolin-1-GFP or flotillin-1-GFP. Twenty-four hours after transfection, cells were infected with C. jejuni, fixed at the designated time points, and processed for immunofluorescence using a staining protocol that distinguishes extracellular from internalized bacteria. Images corresponding to 15 min after infection show colocalization of (A) caveolin-1-GFP (green) (A) and flotillin-1-GFP (green) (B) with intracellular C. jejuni (red). The colocalization of C. jejuni with caveolin-1-GFP (C) or flotillin-1-GFP (D) was quantified at the indicated times after infection as described in Materials and Methods. Results are the means and standard deviation of three independent experiments. For each experiment, at least 100 vacuoles were counted for colocalization with each marker for each time point. In addition, Cos-1 cells were transfected with plasmids expressing caveolin-1-GFP, dynamin II-GFP, or their dominant negative mutants caveolin-1^Y14F-GFP or dynaminII^K44A-GFP (E and F). Twenty-four hours after transfection, cells were infected with C. jejuni, fixed, internalized bacteria were quantified 3 h after infection as described in the Materials and Methods. The values are the mean and standard deviation of three independent experiments and are expressed as a percentage of the control (caveolin-1-GFP and dynaminII-GFP, respectively), which was considered to be 100%. For each experiment the number of intracellular bacteria was determined for at least 50 transfected cells. The difference between the levels of internalization in cells expressing wild type and dominant-negative mutant forms of caveolin or dynamin were significantly different (unpaired t-test p = 0.02 for both sets of samples).